• Korean Journal of Microbiology
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• v.26 no.3
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• pp.197-206
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• 1988

An outer membrane-associated 2-furaldehyde dehydrogenase, catalyzing the oxidation of 2-furaldehyde to 2-furoic acid from Klebsiella pneumoniae was purified to homogeneity and characterized. The enzyme showed its highly specific dependency on $\beta$-$NAD^{+}$. Enzyme activity was monitored during purification by using substrate 2-furaldehyde and coenzyme $\beta$-$NAD^{+}$ by means of high performance liquid chromatography. The outer membrane was successfully collected by the methods of Percoll density gradient ultracentrifugation and ultracentrifugation after preferential solubilization of the membrane with $Mg^{2+}$ and Triton X-100. The enzyme was purified by the series of procedures including extraction of outer membrane protein with EDTA and lysozume, and fractionation by column chromatography on QAE-Sephades Q-50, and subsequently Sephadex G-100. The enzume showed its optimal activity at $85^{\circ}C$, pH 9.5, and in the presence of 1.5% (vol/vol) Triton X-100. The enzyme exhibited a native molecular size of 88,000 by nondenaturing polyacrylamide gel electrophoresis and had an apparent Km of 4.72mM for 2-furaldehyde.